Polypeptides of feline T-cell lymphotrophic lentivirus

ABSTRACT

Purified polypeptides of Feline Immunodeficiency Virus (FIV) are disclosed. The polypeptides of the invention include p10, p15, p26, gp40, gp100 and gp130.

This invention was made with government support under Grant AI-27605awarded by the National Institutes of Health. The government has certainrights in the invention.

This is a continuation of application Ser. No. 08/120,311, filed Sep.13, 1993, now abandoned; which is a continuation of 07/447,810, filedDec. 8, 1989, now abandoned; which is a continuation of 07/348,784,filed May 8, 1989, now abandoned; which is a continuation of 07/293,906,filed Jan. 5, 1989, now U.S. Pat. No. 5,219,725; which is a continuationof 07/279,989, filed Dec. 5, 1988, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to purification of polypeptides from felineT-cell lymphotropic lentivirus (FIV).

FIV is a retrovirus originally isolated from cats which exhibit anAIDS-like syndrome. Pederson et al., 235 Science 790, 1987. The virusbelongs to the same group as the human immunodeficiency virus (HIV), thecausative agent of human AIDS. Pederson et al., describe detection ofantibody to FIV by an immunofluorescent assay, and by Western blots,using virus purified by centrifugation on sucrose gradients in Trisbase, pH 7.4, containing 0.1M NaCl and 1 mM EDTA. A few protein bandswere detected and, although antigenic comparison was not made, thepositions of these bands were tentatively said to correspond to themajor core protein, p24 the gag precursor protein, p55, and theendonuclease protein, p32, of, HIV.

Pederson et al., U.S. Pat. No. 5,037,753 entitled "Feline T-LymphotropicLentivirus" (which is not admitted to be prior art to the presentapplication) describes the results presented by Padarson et al., supra,and states that Western blotting of FIV infected cell lysates yieldedmajor protein bands at approximately 22-26 kD, usually about 24 kD;50-60 kD, usually about 55 kD; and 28-36 kD, usually about 32 kD.

SUMMARY OF THE INVENTION

In a first aspect, the invention features a purified polypeptide havingan epitope of an antigenic polypeptide of FIV. The polypeptide may beglycosylated or unglycosylated. By antigenic polypeptide is meant apolypeptide which is able to raise (with the aid of an adjuvant ifnecessary) an antibody response in cats. The polypeptide may be apolypeptide fragment of at least 5 amino acids, or a polypeptidenaturally occurring in FIV particles. The fragment may be obtained froma naturally-occurring polypeptide, for example, by enzymatic digestionof a naturally occurring polypeptide, or may be produced by isolation orsynthesis of a gene encoding a desired polypeptide and expression ofthat polypeptide within a desired expression system, for example, abacterial, yeast, or mammalian expression system.

By epitope is meant a single antigenic site of an antigenic polypeptide.Such an epitope is recognized specifically by a monoclonal antibody toan antigenic polypeptide of FIV.

By purified is meant that the polypeptide is separated from other cellcomponents with which it naturally occurs, for example, FIVpolypeptides. Preferably, the polypeptide is sufficiently pure to permitits use to prepare a monoclonal antibody to the polypeptide, and evenmore preferably, pure enough to allow the amino acid sequence of thepolypeptide to be determined by standard procedure. Generally, thepurified polypeptide is biologically active in that it is suitable forpreparation of a monoclonal antibody, or is suitable for detection ofnaturally-occurring antibodies within the serum of a cat.

In preferred embodiments, the purified polypeptide has at least 75%amino acid homology to a polypeptide fragment of at least 20 amino acidsobtained from an FIV gag or env polypeptide, most preferably thepurified polypeptide includes an amino acid sequence having at least 75%homology to a whole of a gag or any polypeptide, even more preferably,the purified polypeptide is an entire gag or env amino acid sequence.Examples of gag and env polypeptides include p10, p15, p26, gp40, gp100,and gp130.

In a second aspect, the invention features a method for detecting anantibody to FIV within a sample, including the steps of providing apurified polypeptide as described above, and contacting that polypeptideunder conditions suitable to allow an antibody/polypeptide complex toform between antibodies within the sample and the purified polypeptide,and detecting the formation of such complexes. The presence ofantibody/polypeptide complexes is indicative of antibody to FIV presentwithin the sample.

In a third aspect, the invention features a purified nucleic acidincluding a 50 nucleotide sequence having at least 90% homology with a50 nucleotide sequence naturally occurring in an FIV particle. Bypurified is meant that the nucleic acid is substantially separated awayfrom all of the components with which it naturally occurs, e.g.,polypeptides and other nucleic acids. Preferably, the nucleic acid iscompletely separated from such components, and is a pure solution ofnucleic acid, or is held within a cell in which it does not naturallyoccur, e.g, a bacterial cell, another viral particle or a non-felineeucaryotic cell. By 90% homology is meant that the nucleotide sequenceis identical at at least 45 of the 50 nucleotides.

In preferred embodiments, the nucleic acid encodes a polypeptideincluding an epitope of an antigenic polypeptide of FIV, e.g., anepitope of a gag or env polypeptide of FIV. Most preferably the nucleicacid is carried in an expression vector and can be expressed in abacterial, fungal or other eucaryotic cell, e.g., a mammalian cell.

In a related aspect the invention features purified polypeptideincluding ten or more contiguous amino acids taken from the sequence(using standard letters to represent amino acids)V-Q-S-R-G-S-G-P-V-C-F-N-C-K-K-P-G-H-L-A-R-Q-S-H orP-I-Q-T-V-N-G-V-P-Q-Y-V-A-L-D-P-K-M-V-S or S-V-Q-S-R-G-Q-G-P-V-A-F-N.

Applicants have provided polypeptides suitable for specific detection ofFIV antibodies and thus have allowed accurate detection of infectionwith FIV. Applicants have also provided polypeptides useful forproduction of vaccines to prevent disease caused by FIV in cats.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiment thereof, and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first briefly be described.

Drawings

FIG. 1 is a photograph of the major vital associated proteins of FIVidentified by polyacrylamide gel electrophoresis (PAGE) and stained withCommassie Blue R250 (lane A); molecular weight standards are shown inlane B;

FIG. 2 is a photograph of a Western immunoblot analysis of antibodies toFIV found in serum from cats identified as positive by an ELISA assayfor FIV antibodies;

FIG. 3 is a graphical representation of elution of FIV polypeptidesduring HPLC purification;

FIG. 4 is a photograph of an SDS-polyacrylamide gel afterelectrophoresis and staining with silver, showing purity of various FIVpolypeptides; and

FIGS. 5A-5C show the nucleic acid sequence, and coresponding amino acidsequence, of various portions of cloned FIV nucleic acid.

Antigenic FIV Polypeptides

FIV polypeptide antigens useful in this invention are generallydescribed above. Polypeptides useful in this invention may be purifiedfrom virus isolated as described below and fragments of the purifiedpolypeptides isolated by enzymatic treatment or other standardprocedures. Further, the polypeptides may be synthesized by standard invitro expression systems in which DNA encoding for the FIV polypeptideis cloned and expressed in a bacterial, yeast, or mammalian cellexpression system. Such DNA may be isolated and expressed as describedbelow. The polypeptides may also be synthesized by standard chemicalmethods, for example, the polypeptide segments of various FIVpolypeptides given below can be synthesized. In the following exampleFIV polypeptides were obtained directly from FIV-infected cells and tookthe form of polypeptides naturally occurring in an FIV virus particle.This example is not limiting to the present invention, and those skilledin the art will recognize the many alternative methods for obtainingpolypeptides of this invention. The polypeptides are referred toaccording to molecular weight, thus a polypeptide of 30 kD is termedp30, and a glycoprotein of this weight is termed gp30.

Master seed virus producing cultures were obtained in the form of acontinuous feline cell line infected with FIV isolate #2427 (CRFK-FIV orPetaluma strain) from Dr. Nails Pederson (University of California,Davis, Calif.). The parent cell line is Crandell feline kidney cellpersistently infected with FIV. The cell line was deposited with theAmerican Type Culture Collection on Jul. 13, 1988 and assigned thenumber CRL9761. Applicants and their assignees acknowledge theirresponsibility to replace this culture should it die before the end ofthe term of a patent issued hereon, 5 years after the last request for aculture, or 30 years, whichever is the longer, and its responsibility tonotify the depository of the issuance of such a patent, at which timethe deposit will be made irrevocably available to the public. Until thattime the deposit will be made available to the Commissioner of Patentsunder the terms of 37 C.F.R. §1-14 and 35 U.S.C. §112.

Other virus cultures can be obtained as described by both Pederson etal. references, supra, or by Harbour et al., 122 The Veterinary Record84, 1988. Seed stocks of virus producing cell cultures were obtained byfreeze-downs of FIV-infected master seed cell cultures following atleast 19 post infection passages in culture. Additional seed stocks ofvirus producing cultures were obtained by either infection of thecontinuous feline cell culture with FIV master seed virus or by singlecell microwell cloning of high level FIV producers from the original FIVinfected master seed cell culture. For propogation, master seed virusinfected feline cell cultures were inoculated into tissue cell cultureflasks. Following growth to a confluent monolayer of cells, tissueculture fluid was harvested at intervals of 2-5 days.

Working seed virus was produced by propogation by the master seed cellline permanently infected with FIV. An inoculum was added to tissueculture flasks, in Dulbecco's Modified Eagles medium containing 2 mML-glutamine and 4.5 g per liter/glucose (DME) containing 100 units perml. penicillin and streptomycin and 2 mM glutamine. An inoculum wasadded to tissue culture flasks, incubated, and the spent tissue culturefluid harvested when the cells were grown to confluence. The cells werereleased from the culture vessel with trypsin/EDTA and diluted between1:5 and 1:25 (typically 1:8) in medium. Typically the flasks wereincubated at 36° C.-38° C. for a maximum of 7 days (between 3 and 7days) before fluid and cell harvest. The harvested fluid, including cellmaterial, was centrifuged in a high speed centrifuge (Sorval RC-5B orBeckman J2-21) leading to separation of supernatant and cell pelletmaterial. The cell pellet was discarded, and the supernatant culturefluid used to prepare working virus. The clarified supernatant was made0.5M in NaCl and 4%-10% (usually 7%) in polyethylene glycol (PEG 8000,Sigma). Following overnight incubation at 2° C. -7° C., virus waspelleted (at 13,000×g for 30 min.) a confluent monolayer of cells,tissue culture fluid was harvested at intervals of 2-5 days.

Working seed virus was produced by propogation by the master seed cellline permanently infected with FIV. An inoculum was added to tissueculture fupernatant centrifuged on a 50%/80% discontinuous glycerol stepgradient in 10 mM Tris 300 mM NaCl, 1 mM EDTA at pH 7.6. Centrifugationwas at 100,000×g for 3 hrs. at 4° C. and the FIV viral band at the50%-80% interface collected. The band was suspended in 10 mM Tris, 0.3 MNaCl and 1 mM EDTA and diluted 1:3 in the buffer and repelleted at100,000×g for 1 hr. The resulting pellet was purified virus and wasresuspended in the above buffer and stored at -70° C. The resultingvirus was substantially free from FIV host cell proteins and wascomposed of at least 5% p26 (the major nucleocapsid protein, as measuredby densitometric scans of Commassie Blue 250 stained SDS/PAGE as totalprotein).

Such purified virus may be obtained by other techniques, however,applicants have found that high molecular weight contaminants present invirus preparations may be eliminated by use of the high salt (i.e.,greater than physiological range salt concentration) used in thegradient centrifugation procedure.

Referring to FIG. 1, polypeptides associated with purified FIV wereanalyzed by SDS/PAGE and compared with polypeptides isolated in anidentical manner from the spent culture medium of uninfected cells.Analysis of the Commassie Blue stained gels revealed three majorpolypeptides with molecular weights of about 10, 15, and 26 kD, namedp10, p15 and p26, respectively.

When an ELISA test was performed using disrupted FIV to identify catspossessing polyclonal antibody to FIV polypeptides, and Western blotanalysis then performed on feline sera determined to be positive byELISA, each of the cats had antibodies which reacted with one or morepolypeptides of molecular weight p10, 15, 26, 40 and 65 kD under theconditions used.

Referring to FIG. 2, a standard Western immuno blot was performed asdescribed by Towbin et al., 76 Proc. Natl. Acad. Sci USA 4350, 1979.Briefly, FIV was disrupted with SDS and proteins transferred to a sheetof nitrocellulose. The nitrocellulose sheet was blocked with 30% calfserum, 1% bovine serum albumin (BSA), and 0.05% Tween 20 in Dulbecco'sphosphate buffer saline. The sheets were cut into 0.5 cm strips andincubated with a 1:100 dilution of serum sample in blocking buffer for 2hrs. for 20°-22° C. Strips were repeatedly washed with washing buffer(0.05% Tween 20 in Dulbecco's phosphate buffer saline) and thenincubated with a second antibody (specific for feline heavy and lightchain Ig) horseradish peroxidase conjugate (obtained from Kirkguard andPerry Laboratories Inc. Gaithersburg, Md.). After 1 hr. incubation, thestrips were repeatedly washed with washing buffer and incubated with theprecipitating substrate 4-chloronaphthol for 10 min. The strips werepartially dried and the results interpreted immediately. The serum ineach of the lanes A-G was obtained from various cats infected with FIV.Predominant cross-reactivity is detected with p26 and p15 and to alesser extent with p10. Other proteins of 32, 40, 47 and 65 kD molecularweight are also detected.

Certain vital polypeptides, such as the gag (e.g., p26) antigenicpolypeptides, are abundant in purified viral preparations, others suchas the viral envelope polypeptides (e.g., gp130) tend to be lost duringviral purification, and electrotransfer less efficiently for Westernblot analysis than the gag antigens. Therefore, in order to more readilydetect the viral envelope (env) and the gag precurser polypeptides, FIVcell extracts were labeled with ³⁵ S-methionine and cysteine andexamined by immunoprecipitation (RIPA). Confluent cultures of cellsinfected with FIV were incubated for 30 min. in methionine andcysteine-free Dulbecco's modified Eagle's medium. The cell cultures werethen incubated for 4 hrs. in 8 ml of the same medium containing 100microCuries per ml of ³⁵ S-methionine and ³⁵ S-cysteine (specificactivity 1200 Curies per mM, New England Nuclear Corporation, Boston,Mass.). The radioactive tissue culture fluids were removed and the cellslysed with 5 ml of 10 mM sodium phosphate buffer pH 7.5 containing 100mM NaCl 1% Triton X 100, 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mMphenylmethylsulfonylfluoride, and 100 Kallikren inactivator units ofaprotenin per ml. (Sigma Chemical Co., St. Louis, Mo.). Before use, thecell lysates were clarified by centrifugation 100,000×g for 30 min. andthe pellet discarded. Aliquots of the labelled cell lysates (0.1 ml) and5 μl of serum being tested were mixed in a microcentrifuge tube andincubated for 1 hr. at 37° C. and then overnight at 4° C. The next day,0.2 ml of a 5% suspension of protein A Sepharose CL-4B beads (Pharmacia,Piscataway, N.J.) in 10 mM of phosphate buffer, pH 7.5 containing 100 mMNaCl, 1 % Triton X-100 and 0.1% SDS was added to each tube and mixed for30 min. at 4° C. The antibody/antigen complexes bound to the protein ASepharose beads were collected by centrifugation (2 min. at 20,000×g)and washed 3 times in lysing buffer. The final pellet was resuspended in25 μl SDS/PAGE loading buffer and heated and 100° C. for 3 minutes. TheSepharose beads were removed by centrifugation and the supernatantapplied to a PAGE. Gels were processed for fluorography usingenlightening™ (New England Nuclear Corporation, Boston, Mass.) andexposed at -70° to Kodak XR-5 film. Sera from experimentally infectedcats recognize polypeptides of 15, 22, 36, 40, 47, 110 and 130 kD.Although there were some quantitative and qualititative differences allcats appear to mount a response to p22, gp40, gp47 and gp130.

In order to determine which of the polypeptides identified by RIPA-PAGEanalysis were related to the major internal structural protein, p26,RIPA-PAGE analysis was carried out using monoclonal antibodies whichreacted with p26 as determined by Western blotting. This monoclonalimmunoprecipited proteins p47, p36, p22 and p15. High molecular weightpolypeptides (130 kD) of FIV which were not detected by the p26 specificmonoclonal antibody, were identified by RIPA-PAGE. A protein ofmolecular weight 100 kD was also detectable utilizing serum antibodiesobtained from some infected cats.

In another example, antigenic glycopeptides of FIV can be obtained asfollows. Actively growing CRFK FTLV infected cells were scraped fromroller bottles, gently washed with phosphate-buffered saline (PBS), andpelleted. The cell pellet was gently resuspended in 10 mM sodiumphosphate, pH 7.2, at a ratio of 1 ml buffer to 0.1 ml of cell pellet.This suspension was incubated on ice or refrigerated for 5-10 min.,vigorously vortex mixed for 30 seconds, and four volumes of PBS with 1mM PMSF added. The mixture was then vigorously homogenized for 90-120seconds with a Brinkmann Homogenizer PT10/35 with a PTA 20 generator.

The resulting homogenate was clarified for 20 minutes at 5,000×g. Thesupernatant fraction was discarded and the cell membrane pelletresuspended in PBS+0.2% Triton X-100 at a ratio of 2.5 ml buffer to 0.1ml original cell pellet. The mixture was then vigorously homogenized for90-120 seconds with a Brinkmann Homogenizer PT 10/35 with a PTA 20generator. The resulting homogenate was clarified at 100,000×g for 1 hr,the supernatant decanted off and batch bound overnight at 20°-23° C. onPharmacia Lentil Lectin Sepharose 4B at a ratio of 6 ml of resin to 5 mlof original cell pellet.

The Lentil Lectin Suspension was poured through a column, the resincollected, and washed with 15 column volumes of PBS+0.2% Triton X-100.The glycoproteins were then eluted from the resin by subjecting theresin to 5-10 column volumes of PBS+0.2% Triton X-100+200 mM methyl α-Dmannopyranoside, collecting fractions of 1 column volume/tube.

The isolation of glycoproteins was verified by 9% SDS-PAGEelectrophorisis, and checked using ³⁵ S-radiolabeled cell preparationsin conjunction with RIPA data.

Further purification of viral glycoprotein from host cell glycoproteinincludes use of HPLC, or of a polyclonal or monoclonal antibody foraffinity chromotography.

EXAMPLE 1: gag Polypeptide Purification

Isolated virus (250-500 microliters) was combined with two volumes of 6Mguanidine hydrochloride, pH 3 (adjusted with 20% trifluoro acidic acidin water). The mixture was vortexed and incubated at 37° C.-40° C. in awater bath for 20-25 minutes. The incubated solution was filteredthrough a pre-wetted (100 microliters of 6M guanidine hydrochloride, pH3) 0.45 micron gel aquadisk filter (No. 4184) and the filter rinsed with100 microliters of 6M guanidine hydrochloride pH 3. The filtered samplewas loaded onto an HPLC column for purification.

The HPLC system consisted of a Beckman HPLC having three 110 V pumps, a421 controller, a 166 variable wavelength detector, a 427 integrator, a210A injector with dynamic mixer, and a 1000 microliter sample. Thecolumn was a Waters radial compression cartridge held in an RCM-100column holder with a modified inlet connector (Waters MicroBond-A-PakFenile 10 MU 8 mm ×10 cm cartridge No. 85722 with a guard pak resolve CNcartridge, No. 85826). The system was set such that two levers werecompressed and the pressure was in the mid-yellow zone.

Purification was by a multi-step gradient from aqueous 0.1%trifluoroacetic acid (v/v solvent A) to aceto nitrol containing 0.1%trifluoroacetic acid (v/v, solvent B).

Fractions were collected and condensed by a Savant Speed Vac to removeall solvents to a final volume ranging from 50-100 microliters. Thecondensed fractions where neutralized by addition of 200-300 microlitersof 50 mM or 100 mM sodium phosphate buffer, pH 7.2. The bufferedfraction pHs were then checked with pH paper and, if the pH was stillbelow 6, 1N NaOH added until the pH was brought within the range of6.5-7.5. The neutralized fractions were frozen at -20° C. until use.

Referring to FIG. 3, the printout from an HPLC column described above isshown. The flow rate was at 1 ml per minute starting with 100% ofsolvent A. After 15 min. the solvent was changed to contain 26% solventB, 10 min. later to 31% solvent B, 12 min. later to 37.5% solvent B, 5min. later to 40% solvent B, 6 min. later to 43% solvent B, 8 min. laterto 45% solvent B, 15 min. later to 60% solvent, and 20 min. later to100% solvent B. Peaks corresponding to p10, p15, and p26 are indicatedin FIG. 3. Fractions containing these peaks were collected.

Referring to FIG. 4, fractions corresponding to p10, p15 and p26 wererun in an SDS-polyacrylamide gel containing 15% bis-acrylamide at 70 V.The gel was stained with silver using a Biorad Silver Staining Kit161-0443. The isolated fractions corresponding to p15, p10, and p26 wereessentially homogeneous solutions of the FIV polypeptides.

The isolated FIV polypeptides were analyzed by standard technique fortheir amino acid sequence, yielding the following results (? indicatesuncertainty in the actual amino acid, or no knowledge at all).

    ______________________________________                                        p26                                                                           Number  1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19-20                    Amino Acid                                                                            P-I-Q-T-V-N-G-V-P- Q- Y- V- A- L- D- P- K- M- V- S                    p10                                                                           Number  1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-16-17-18-19-                      Amino Acid                                                                            V-Q-S-R-G-S-G-P-V- C- F- N- C- K- K- P- G- H- L-                      Number  20-21-22-23-24                                                        Amino Acid                                                                            A- R- Q- S- H                                                         P15                                                                           Number  0-1-2-3-4-5-6-7-8-9-10-11-12-13                                       Amino Acid                                                                            S-V-Q-S-R-G-Q-G-P-V- A- F- N- ?                                       ______________________________________                                    

In order to determine if the purified polypeptide is useful in thisinvention, that is, whether the polypeptide is antigenic, any standardprocedure can be used. For example an ELISA test can be performed usinga polyclonal antibody from cat serum to determine whether thepolypeptide is cross-reactive. Alternatively, the polypeptide can beinjected with or without an adjuvant, into an animal, e.g., a mouse, todetermine if it causes antibodies to be raised to it. These polypeptidesare useful for production of vaccines to prevent FIV-caused diseasesymptoms and FIV infection in cats. These vaccines are produced bystandard procedure. Preferably the gag or env polypeptides are providedin a standard inoculation medium and injected intravenously,intrarterially or otherwise into a cat at a level of 1-100 μg/kg animalat intervals of 3-4 weeks until immunity to FIV is produced.

FIV Monoclonal Antibodies

Antibodies to FIV polypeptides are useful aids for identification of apurified polypeptide (as described above), and for purifyingpolypeptides. They are also useful to determine the antigenicity of anypolypeptide. An example of preparation of useful antibodies follows.These antibodies are monoclonal antibodies which allow specificdetection and purification of either individual or a small number of FIVpolypeptides.

Balb/CJ (Jackson Labs) mice were immunized with an initial injection of50 micrograms of FIV virus (prepared as described above) per mouse mixed1:1 with Difco Bacto adjuvant complete. After two weeks a boosterinjection of 100 micrograms of FIV virus was injected into each mouseintravenously without adjuvant. Three days after the booster injection afusion was performed with mouse myeloma cell lines FO or p3X63-Ag8.653.Mid log phase myeloma lines were harvested on the day of fusion andchecked for viability. The cells were spun at 300×g for 8 min.,separated from the growth medium, and resuspended in serum free DME.

For fusion, an FIV-inoculated mouse was killed by cervical dislocationand the spleen aseptically removed. The spleen was washed three times inserum free DME and placed in a sterile Petri dish containing 20 mls ofcomplete medium (DME containing 20% bovine fetal serum, 100 units perml. of penicillin and streptomycin, and 1 mM sodium pyruvate). Torelease cells, the spleen was perfused with a 23 gauge needle.

Cells were placed in a 50 ml conical centrifuge tube and pelleted at300×g for 8 min. The pellet resuspended in 5 ml of 0.17M ammoniumchloride and placed on ice for 8 min. 5 ml of bovine fetal serum (20%)was added and the cells pelleted again at 300×g for 8 min. Afterresuspension in 10 ml DME the cells were counted and the spleen andmyeloma cells mixed in a ratio of 3:1. The cell mixture was pelleted at200×g for 10 minutes, the supernatant decanted, and the pellet allowedto stand for 5 min. Over a period of 1 min., 1 ml of 50% PEG (PEG 1500mixed 1:1 with Hepes pH 8.1) at 37° C. was added. After 1 min.incubation at 37° C., 1 ml of DME was added over a period of another 1min. and then a second 1 ml of serum free medium added over a period of1 min. Finally, 10 mls of DME was added over a period of 2 min., thecells pelleted at 200×g for 8 min., and the pellet resuspended incomplete medium containing 0.016 mM thymidine, 0.1 mM hypoxanthine, 0.5micromolar aminopterin, and 10% hybridoma cloning factor (1×HAT). Thecells were plated into 96-well plates.

After 3, 5 and 7 days half of the medium in the fusion plates wasremoved and replaced with fresh 1×HAT. After 11 days the hybridoma cellsupernatant was screened by an ELISA test. In this test, 96 well plateswere coated with FIV virus by standard technique. One hundredmicroliters of supernatant from each well was added to a correspondingwell on a screening plate and incubated for 1 hr. at 20°-22° C. Afterincubation, each well was washed three times with distilled water and100 microliters of a horseradish peroxidide conjugate of goat anti-mouseIgG (H+L), A, M (1:1500 dilution) was added to each well and incubatedfor 1 hr. at 20°-22° C. After three washes with distilled water, thesubstrate OPD/hydrogen peroxide was added and incubation continued forfive to fifteen minutes. One hundred microliters of a stop solution (1Mhydrochloric acid) was then added and the absorbance at 490 nm read.Cultures which had an optical density reading greater than the controlwells were removed to 2 cm² culture dishes, with the addition of normalmouse spleen cells in 1×HT medium. After a further three days all of the2 cm² cultures were rescreened for antibody and those testing positiveagain were cloned by limiting dilution. The cells in each 2 cm² culturewere counted and cell concentration adjusted to 1×10⁵ cells per ml. Thecells were diluted in complete medium and normal mouse spleen cells atconcentrations of hybridoma cells of 5, 10 and 50 cells per ml added.The cells were plated into 96-well plates for each dilution. After 10days the cloning plates were screened for growth. About 37% of all wellsshowed growth. The growth-positive wells were screened for antibody andthose testing positive expanded to 2 cm² cultures and provided withnormal mouse spleen cells. The cloning procedure was repeated 2 timesuntil a stable antibody-producing hybridoma was obtained. At this pointthe cell culture was expanded from 2 to 9 to 75 to 150 cm² culturevessels, at which point ascite production could be commenced.

For ascites production, pristane primed IRCF1 female mice were used. 0.5ml of pristane was injected intraperitoneally (IP) to each mouse, andthe mouse allowed to rest for 10-60 days. At this time 4.5×10⁶ cellswere injected IP into each mouse and ascites formed in 7-14 days.Ascites fluid was harvested with a pasteur pipette through a hole in theperitoneum.

Antibodies to glycoproteins can also be isolated and detected. Inparticular, antibodies to two glycoproteins of molecular weight 40 kD(gp40) and 130 kD (gp130) which are detected using PAGE and RIPArespectively.

Monoclonals useful in this invention for purification and identificationof specific polypeptides of FIV include those which are specific for FIVand form a sufficiently strong interation with an FIV epitope, and anFIV antigen, to be useful in an immunoassay, for example, an ELISA, todetect an FIV antigen. In order to determine which of the abovemonoclonal antibodies are useful in this invention one main test wasused. This entailed determination of whether the monoclonal antibody canbind FIV antigen and be detected with a conjugate of polyclonal antibodyto FIV (an ELISA test, described above).

Another test is to perform a Western blot to determine whether themonoclonal antibody has good reactivity with one or more FIV antigens.Generally, those monoclonals which show poor reactivity, that is,produce faint bands on the Western blot, are not suitable in thisinvention. Yet another test involves radioimmunoprecipitation assay(RIPA) where FIV virus labeled with ³⁵ S-methionine is reacted with amonoclonal antibody to form within immunoprecipitate, and theimmunoprecipitate run in a SDS-PAGE and autoradiographed to detect thelabelled proteins. This analysis determines which of the monoclonalantibodies is able to detect precursor FIV antigenic polypeptides andnot just mature polypeptides.

Antibody Detection

The above antigenic polypeptides can be used to detect naturallyoccurring antibodies produced by cats. Such detection can be anystandard immunoassay procedure, for example, by an ELISA test, asdescribed above. One example of such a test follows. This example is notlimiting to the present invention and those skilled in the art willrecognize that any of many other standard procedures can be used.

EXAMPLE 2: Antibody assay

Materials required to perform this assay include 96 well flat bottommicrotiter strips coated with a solution containing the appropriate testantigen (e.g., p26, p15, or p10). The test wells were coated with 100 μlof a solution containing 0.15 micrograms antigen in 0.25 molar sodiumcitrate, pH 7.5. The wells were covered with parafilm, incubatedovernight at 4° C., and tapped until dry. The antigen was thenovercoated by adding 200 μl 1% BSA in 0.25 molar sodium citrate,incubating at room temperature (20°-25° C.) for 1 hour, and tapping thewells dry. 200 microliters of 7.5% sucrose in 0.25 molar sodium citratewas then added to each well and incubated at room temperature for 30minutes. The resulting strips were used immediately, or dried undervacuum for 6 hours at room temperature for later use.

Assays were performed by adding 100 μl of feline serum sample (positivecontrol, negative control, or test sera) diluted 1 to 100 in Dulbecco'sPBS containing 0.1% Bovine serum albumin, 30% calf serum, and 0.05%Tween-20 (Sigma Chemical, St. Louis, Mo.) to a well, incubating at roomtemperature for 30 minutes, and tapping the well dry. The wells werewashed immediately two times with Dulbecco's PBS containing 0.05%Tween-20, tapping the wells dry after the second wash.

100 μl of a solution containing antibodies to feline immunoglobulin wasthen added. These antibodies were conjugated to an indicator enzyme(e.g., alkaline phosphatase) and then dissolved in a solution of 50%fetal calf serum, 0.05% Tween-20 in 0.05M Tris-HCl, pH 7.6. The wellswere incubated at room temperature for 30 minutes, tapped dry, and thenwashed two times with Dulbecco's PBS containing 0.05% Tween-20.

A solution containing 0.1% 3,3',5,5' Tetramethylbenzidene in 40%Glycerol and 60% methanol was mixed with an equal volume of a solutioncontaining 22.82 grams dibasic potassium phosphate, 19.2 grams citricacid and 1.34 milliliters 30% hydrogen peroxide solution per liter. Onehundred microliters of this solution was added to each well, and thenincubated at room temperature for 15 minutes. At the end of theincubation period, 100 μl of 0.25% hydrofluoric acid was added to eachwell. The optical density at 650 nanometer of the solution in each wellwas then determined with a microtiter plate reader. An immune responseto p10, p15 and p26 was detectable either following experimentalinfection of a cat with FIV, or in feline sera possessing antibodies toFIV.

FIV Nucleic Acid

FIV nucleic acid is useful for production of large amounts of FIVpolypeptides, or fragments thereof, and also for detection of homologousnucleic acid in vivo, using standard techniques. There follows anexample of cloning of FIV viral DNA. The specific FIV strain chosen isnot meant to be limiting in this invention and those skilled in the artwill recognize that equivalent nucleic acid may be isolated by use ofthe cloned sequences which are provided as specific deposits, or bytechniques similar to those described in this example.

A Crandal feline kidney cell line productively infected with FIV strain2428 (Pentaluma isolate) was used as a source of unintegrated viral DNA.The unintegrated vital DNA was prepared by Hirt extraction andCsCl-ethidium bromide centrifugation to resolve linear and supercoiledviral DNA. (Hirt, 26 J Mol. Biol. 365, 1967; Canaani et al., 282 Nature378, 1979).

The supercoiled viral DNA was used to construct libraries which containoverlapping viral DNA sequences. The procedures used to construct theselibraries were similar to those described by Maniatis et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Lab, Cold SpringHarbor, N.Y.) and Glover, DNA Cloning, Vol. 1, A Practical Approach, IRLPress, Washington, D.C.) and are familiar to those of ordinary skill inthe art.

Two vital DNA libraries were generated by cleavage of supercoiled vitalDNA by one of two restriction endonucleases. Partial cleavage by therestriction endonuclease RsaI, which recognizes the DNA sequence5'GTAC3', or cleavage by the restriction endonuclease HaeIII, whichrecognizes the DNA sequence 5'GGCC3', of the supercoiled viral DNAgenerates two sets of blunt-ended DNA molecules whose sequences overlap.The blunt-ended DNA molecules were then treated with EcoRI methylase,according to the manufacturer's directions, to modify the 3' adenineresidue of the EcoRI recognition sequence 5'-GAATCC-3'. Methylation atthis site inhibits the cleavage of treated DNA by the restrictionendonuclease EcoRI. The methylated DNA molecules were then ligated tolinker DNA molecules which contained a cleavage site for the restrictionendonuclease EcoRI. The linker containing DNA molecules were thentreated with the restriction endonuclease EcoRI to generate moleculeswhose termini were compatible with the EcoRI cloning site in therecombinant DNA phage vector λ ZAP™ (Stratagene, La Jolla, Calif.).Linker fragments resulting from this cleavage were separated from thelarge DNA molecules by size separation on a quickspin column(Boehringer, Mannheim, Indianapolis, Ind.). The DNA molecules were thenligated into the EcoRI cleaved λ ZAP vector using T4 DNA ligase (NewEngland Biolabs, Beverly, Mass.). Ligated DNA molecules were thenpackaged into phage using Gigapack gold (Stratagene, La Jolla, Calif.).Viable phage from the packaging reaction were then amplified byinfecting BB4 cells (Stratagene) and harvesting plate lysates of thoseinfected cells in order to obtain a stock of recombinant λZAP clones.

Because the insert DNA of the recombinant λZAP clones contain hostcellular DNA as well as FIV proviral DNA sequences, it was necessary toscreen each library with an FIV DNA probe that contains a readilydetectable label. Such a probe was made from RNA isolated from FIV. Aradioactive complementary DNA was synthesized from total viral RNAessentially as described by Maniatis et al. 1982, supra except thatselection of poly A-containing RNA was not performed, and methymercurichydroxide was omitted from the protocol.

The bacteriophage libraries were plated at a density of 10,000bacteriophage per 150 mm dish. They were then screened by hybridizationof the radioactively labeled probe to phage DNA which was immobilized onnitrocellulose filters (Maniatis, et al., 1982 supra). Each hybridizingbacteriophage plaque was then picked, replated, and hybridized asdescribed, until a single well was isolated which contained a λZAPrecombinant clone. XL-1-Blue cells (Stratagene) were then infected withrecombinant λZAP phage, and plasmids containing the insert DNA wereobtained following superinfection with R408 helper phage according tothe manufacturer's directions (Stratagene, La Jolla, Calif.). Thisprocedure also provides both recombinant plasmids (which can be isolatedfrom the cell) and single stranded phage stock which can be isolatedfrom the medium for DNA sequence analysis.

The above recombinant plasmids were analyzed for inserts by preparingplasmid DNAs from overnight culture of bacteria replicating theseplasmids as follows. One and one half ml. of an overnight culture wasplaced in a microcentrifuge tube and spun for four minutes at 4000×g.The supernatant was removed and the tube respun for four minutes at4000×g. The supernatant was removed and the tube respun for a fewseconds, and residual liquid removed carefully with a pasteur pipet. Thebacterial pellet was then thoroughtly resuspended in 200 microliters ofa solution contaiing 8% sucrose, 50 mm EDTA, 5% Triton X-100 and 50 MMTris/HCl, pH 8.05. 20 microliters of a lysozome solution at aconcentration of 10 milligrams lysozyme per milliliter in 10 millimolarTris/HCl, pH 8 and one millimolar EDTA was then added, mixed, and themixture was incubated at 4° C. for 15 minutes. The solution containingbacteria was then placed in a boiling water bath for 90 seconds. Themixture was chilled on ice, and spun in a microfuge in the cold for 10minutes at 11,000×g. The pellet was carefully removed with a glasspipet. Ice cold isopropanol (200 microliters) was then added, thesolution thoroughly mixed, and incubated at -20° C. for 5 minutes. Thechilled solution was centrifuged at -20° C. at 11,000×g for 10 minutesto pellet the plasmid DNA. The supernatant was carefully removed and thepelleted DNA briefly air dried. The DNA pellet was then dissolved in 100microliters of sterile double distilled water. Plasmid DNAs thusisolated were anlayzed for inserts by restriction enconuclease cleavageand electrophoresis in 0.8% agarose gels (Maniatis et al., 1982 supra).

Standard dideoxy sequence analysis was performed on the recombinant DNAcontaining clones. Single stranded phage were isolated from the mediaused to propagate cells containing the bluescript plasmid using themethod generally described in the M13 dideoxy sequencing manualpublished by Bethesda Research Laboratories (Gaithersburg, Md.). Anumber of clones were sequenced and analyzed by this method. Sequenceinformation for clones termed 10CX, 2BY, and R5X is presented in FIG.5a, b, and c along with the putative translated amino acid sequence foreach clone. These amino acid sequences show homology with the amino acidsequence of the envelope gene of equine infectious anemia virus, alentivirus, immunologically closely related to FIV.

Nucleic acid probes derived from the 2BY DNA sequence hybridize to DNAisolated from FIV infected but not uninfected cells. These probes can beused to isolate other FIV genes from other strains and can be expressedby standard procedures to provide the purified polypeptides describedabove.

Deposit

Strains 10CX, 2B4 and R5X have been deposited with the ATCC (12301Parklawn Drive, Rockville, Md. 20852) and assigned numbers 67937, 67938,and 67939, respectively.

Applicants' and their assignees acknowledge their responsibility toreplace these cultures should they die before the end of the term of apatent issued hereon, 5 years after the last request for a culture, or30 years, whichever is the longer, and its responsibility to notify thedepository of the issuance of such a patent, at which time the depositswill be made irrevocably available to the public. Until that time thedeposits will be made available to the Commissioner of Patents under theterms of 37 CFR Section 1-14 and 35 USC Section 112.

Other embodiments are within the following claims.

We claim:
 1. Purified and isolated FIV gp40.
 2. Purified and isolated FIV gp100.
 3. Purified and isolated FIV gp130. 